From Priming to Therapy: Leveraging New Insights in T Cell Activation

2.1. Integration of Classical Signals, CD4 Help, DC Licensing and Conceptual Challenges Optimal priming of CD8 T cells requires the integration of three signals (i) TCR engagement through recognition of peptide–MHC class I complexes, (ii) costimulatory signaling via CD28–CD80/86 interactions, and (iii) inflammatory cues, such as IL-12 or type I interferons (IFN-I) [14,16]. Following this initial activation, CD8 T cells were believed to differentiate autonomously—a concept termed “CD8 T cells on autopilot” by Michael Bevan [17]. However, evidence from tumor models challenged this view, revealing that the full expansion and differentiation of CD8 T cells into functional CTLs requires an additional component: CD4 helper T cell support [13,18,19]. In vitro studies identified two critical helper signals provided by CD4 T cells: interleukin-2 (IL-2) and CD40 ligand (CD40L) [13]. In vivo, effective help required CD4 and CD8 T cells to recognize the same antigen presented by the same dendritic cell (DC) [20]. This presented a significant conceptual challenge: how can the immune system coordinate productive interactions among three distinct and rare cell types—antigen-specific CD4 T cells, CD8 T cells, and appropriate DCs—within the constrained timeframe of an immune response? Key insights into this process emerged with the advent of intravital microscopy, dramatically enhancing our understanding of T cell priming dynamics. This technique revealed how rapidly T cells migrate, how they navigate the lymph node microenvironment, and how many T cells a single DC can engage per hour. When antigen-specific T cells encounter cognate peptide-presenting DCs, they typically form stable interactions lasting up to 24 h, after which they disengage and begin to proliferate. However, considering the extremely low precursor frequencies of naïve CD4 and CD8 T cells (estimated at 1 in 10⁵–10⁶), it became evident that stable tri-cellular (CD4–CD8–DC) interactions are statistically unlikely. This led to hypotheses involving chemoattractant signaling to promote localized cell clustering. Yet, the concept of stable three-cell interactions was ultimately set aside when studies demonstrated that CD4 and CD8 T cells are typically primed by distinct DC subsets following viral infections [20,21]. Moreover, the timing of their activation is often asynchronous: for instance, CD8 T cells are activated earlier than CD4 T cells in vaccinia virus infections, whereas the reverse is observed in HSV infection models [20,21]. An explanation to this paradox came from the work of Matzinger’s group, who showed that CD4 T cells can “license” DCs through CD40L–CD40 interactions. Once licensed, these DCs are empowered to prime CD8 T cells subsequently, allowing for sequential rather than simultaneous interactions [22]. This model also explained why CD4 depletion profoundly impairs anti-tumor CD8 T cell responses, yet has minimal effects on primary antiviral responses. In viral infections, the presence of PAMPs and high levels of inflammation can compensate for the absence of CD4 help by directly activating DCs. However, CD4 T cell help remains essential for effective memory formation even in viral settings. Multiple studies have since confirmed the critical role of CD40L-mediated DC licensing, which is carried out exclusively by the DC1 subset. The emerging model can be summarized as follows: CD4 and CD8 T cells are primed independently through prolonged interactions with antigen-bearing DCs. After this priming phase, both populations resume motility, allowing CD4 T cells to interact with DC1s, delivering CD40L-mediated licensing signals. Licensed DC1s subsequently engage CD8 T cells, delivering the necessary secondary signals for full CTL differentiation. But how does IL-2 fit into this picture? How can paracrine signals be transmitted effectively if the cells involved are actively travelling through the lymph node? Another unresolved issue emerged: depletion of DC1 during vaccinia virus infection significantly impaired the formation of memory CD8 T cell responses, yet had minimal impact on the acute effector phase. If DC licensing is indeed restricted to the DC1 subset, how can this be reconciled with the observation that CD4 T cells are critical for driving effector responses in tumors, but appear largely dispensable during the primary response to viral infections? 2.2. Solving the Conundrum—A Second Priming Phase in a Specialized Niche Our recent findings solved this conundrum by detailed investigation of later events during CD8 T cell priming. Using intravital microscopy on days 2 and 3 post vaccinia virus infection, we accessed deeper lymph node regions. We discovered that CD8 T cells regained their responsiveness and reengaged with DC for a second time for several hours. After the CD8 T cells were activated for the first time (first phase), they proliferated and upregulated CXCR3, allowing them to access the secondary subfollicular niche and form long-lasting interactions with DC [23]. Once CD8 T cells re-cluster with DC, they obtain additional TCR stimulation and receive IL-2 from CD4 helper T cells that transiently stop at this niche. By developing a novel mouse model that allowed for selective depletion of CD4 helper T cells while sparing regulatory T cells, we found that CD4 help is essential for CD8 T cell effector generation during viral infections. Thus, the delivery of IL-2 by CD4 T helper cells requires their concomitant, yet transient, presence at the site of DC-CD8 T cell interaction. Notably, the presence of DC1 is dispensable for the relay of IL-2 and thus CD8 T cell effector generation [23]. Importantly, CD4 T cells at the second phase of priming delivered their help predominantly to high affinity T cells, as CD8 T cells with low affinity could not access the secondary niche. As a consequence, they did not efficiently expand to form effector cells one week post infection [23]. In summary, while the first priming phase activates a broad range of antigen-specific CD8 T cells, the second priming phase selectively expands high affinity CD8 T cells but also orchestrates DC-licensing and thus optimal memory formation. To promote these events, activated CD8 T cells re-cluster with DC to receive an additional round of TCR signaling as well as IL-2 from CD4 T helper cells. Subsequently, these high affinity T cells efficiently differentiate into effector cells with cytotoxic properties. These results raise an important question: If optimal effector T cell differentiation and selection during the priming process requires specialized niches, does a similar requirement exist during chronic activation, such as in cancer or unresolved infections? Available data suggest that optimal CD8 T cell effector differentiation during chronic infection requires continuous CD4 T cell help, not only during the initial priming phase but also throughout the response [24,25,26,27]. We hypothesize that the fundamental principles during chronic conditions do resemble those of priming—such as CD8 T cell arrest and transient interactions with CD4 T cells to enable paracrine signaling—yet important details may differ, including the participating cell types and their phenotype as well as the local cytokine milieu. Indeed, CD4 T cells have been shown to produce IL-21, rather than IL-2, to drive the differentiation of the cytolytic CX3CR1 effector population during chronic viral infection. Currently, the spatiotemporal dynamics of IL-21 delivery to CD8 T cells in this setting and the precise composition of the local niche remain to be elucidated but could provide important new angles for therapeutic intervention. The situation in chronic infection differs from that in the tumor context, where the extent and nature of CD4 T cell help vary considerably between tumor entities. An especially intriguing aspect is that, in tumors, CD4 T cells seem to provide help not only in the lymph node but also directly within the tumor microenvironment—the effector site itself [28]. The precise role of CD4 T cell help in the tumor setting remains incompletely understood, but it seems to contribute to the complete differentiation of CD8 effector T cells and support their survival [29,30]. However, the quality of such help could be limited due to suppressive signals in the tumor microenvironment. Thus, there is likely a complex interplay between dendritic cells, metabolic factors, local suppressive cues and stromal cells that critically shape the CD8 T cell differentiation within the tumor niche. Following the initial priming of CD8 T cell, if a subsequent second signal occurs at the effector site, it confines these cells within the tumor tissue, leading them towards a systematic loss of function resulting in exhaustion [31]. In line with this, recent spatial data have highlighted spatially coordinated interactions between DC1-CD8 T cell subpopulations within tumor and how they might shape the further differentiation of CD8 T cells [32]. Another lung carcinoma model study shows the importance of efficient TCR engagement with DCs to form and maintain the stem-like population to provide anti-tumor immunity [33]. Additionally, the presence of metabolites like methionine could shape the differentiation of T cells following TCR engagement, highlighting the role of microenvironment [34]. Multiple factors influencing CD8 T cell differentiation in the tumor and its local microenvironment have been identified, but their precise mechanism and coordination remain fully understood. 2.3. Cyclical Priming of CD8⁺ T Cells and the Path to Optimal Function or Dysfunction Hitherto, it was believed that in acute infections, CD8 T cells cluster with antigen presenting cells just once to get activated, after which they proliferate without the need of further stimulation [17,35,36]. However, our recent study about the later events in T cell priming showed that a second activation step is crucial for optimal CD8 T cell response. It is thus conceivable that CD8 T cells go through multiple cycles of TCR de- and re-sensitization and subsequent activation, including migrational arrest and retention within specific niches. The duration of these cycles could differ depending on TCR affinity, where high-affinity CD8 T cells undergoing shorter cycling times. This raises important questions with potential therapeutic implications: If CD8 T cell priming occurs in cycles, at what point does this process shift into exhaustion? Is this transition driven purely by quantitative factors (e.g., number of cycles) or also by qualitative changes in the activation environment? If so, what are these factors, and can they be therapeutically targeted? During persistent infections, the continuous activation of CD8 T cells favors a hierarchical loss of function, leading towards functionally exhausted populations rather than robust effectors. This shift is marked by high expression of inhibitory markers like PD-1, Lag3, Tim-3 and lower cytokine and chemokine polyfunctionality [11,37]. In addition to continuous antigen stimulation, the lack of CD4 help and suppressive signals from inhibitory molecules contribute to the dysfunction [11]. These data support a quantitative model in which CD8 T cell dysfunction progressively increases with successive activation cycles and ultimately becomes fixed. However, the process of T cell exhaustion appears to differ markedly between chronic infection and cancer. In the tumor context, the induction of exhaustion and T cell dysfunction has been observed to be initiated during the initial contact between T cells and DC presenting tumor antigens [38]. Inflammatory cues that activate DC and T cells are an important factor here. It will therefore be crucial to distinguish tumors according to their inflammatory environments, both within the tumor itself and in associated lymphoid structures, to enable the development of tailored therapeutic strategies. In summary, T cell activation proceeds in cycles during both acute infection and chronic stimulation. The surrounding microenvironment plays a decisive role in determining whether CD8 T cells undergo effective differentiation or progress toward dysfunction. Defining the precise localization, cellular composition, and molecular principles of these environments will be essential for optimizing CD8 T cell activation and advancing immunotherapy.